In 1999, the synthesis, extraction, and biological screening of organic compounds was estimated to account for over one-quarter of the 24 billion dollars spent on pharmaceutical research and development. Undoubtedly, much of this expense results from inefficiencies in the laboratory and from failed compounds. Contributions that help reduce the cost of lead compound identification, with respect to both financial and time savings, are desirable.
Dynamic combinatorial chemistry offers the advantage of synthesizing a multitude of compounds under equilibrating conditions with simultaneous receptor binding directing the dynamic equilibrium. In practice, the process has several limitations. First, the chemistry which may be employed as a scrambling mechanism (e.g., during self-assembly or otherwise) is limited by target molecule compatibility. Any type of chemistry that can modify the target may lead to a false hit, and render any ligand identified by the assay suspect. In nucleic acid directed libraries, for example, free radical generating and methylating reagents must not be employed. Reactions catalyzed by extremes of pH are also undesirable. Obviously, any limitations on the chemistry involved in this approach detracts from its utility, so it is advantageous to develop a means that enables the use of the maximum number of scrambling reactions. Second, binding assays are typically done in aqueous solution. However, many organic transformations are incompatible with water. More importantly, many organic transformations are reversible and could be used to generate dynamic diversity. Unfortunately, they would not be useful if they could not be performed in aqueous solvent. These two drawbacks present significant limitations to the overall power of dynamic combinatorial chemistry.
The present invention is directed to overcoming the above-identified deficiencies in dynamic combinatorial chemistry and rendering the process of lead compound identification much more efficient.
One aspect of the present invention relates to a reaction vessel which can be used to identify ligands, which are reaction products of a combinatorial library, that bind to a particular target molecule. The reaction vessel includes a first member defining a first chamber, the first chamber including an organic solvent and a plurality of reactants which form a combinatorial library of products; a second member defining a second chamber, the second chamber including a target molecule and an aqueous solvent immiscible in the organic solvent; and a semipermeable membrane separating the contents of the first chamber from the contents of the second chamber, wherein said semipermeable membrane is permeable to one or more products of the combinatorial library of products.
According to one embodiment, the reaction vessel, absent any reactants, includes: a first reaction container including (i) a body having an open end and (ii) a cap secured about the open end to define a first reaction chamber; and a second reaction container which is sized and configured for placement within the first reaction chamber, the second reaction container including (i) a body having an open end, (ii) a semipermeable membrane positioned over at least a portion of the open end, and (iii) a cap having an aperture therethrough, which cap is secured about the open end with the semipermeable membrane covering the aperture to define a second reaction chamber.
According to a second embodiment, the reaction vessel, absent any reactants, includes: a first reaction container including a body having an open end and defining a first reaction chamber; a second reaction container including a body having an open end and defining a second reaction chamber; and a cap element including (i) a first portion secured about the open end of the first reaction container and having an aperture therethrough, (ii) a second portion secured about the open end of the second reaction container and having an aperture therethrough, and (iii) a semipermeable membrane positioned between the first and second portions and obstructing communication between the apertures thereof.
A further aspect of the present invention relates to a method of identifying a ligand having affinity for a target molecule. This method includes the steps of providing a dual-chambered reaction vessel including first and second containers defining first and second chambers, respectively, which are separated by a semipermeable membrane, with the first chamber including an organic solvent and a plurality of reactants which form a combinatorial library of products, the second chamber including an aqueous solvent immiscible in the organic solvent and, optionally, a target molecule, and the semipermeable membrane being permeable to one or more products of the combinatorial library of products; and identifying any products present in the second chamber at higher concentration while the target molecule is present than without.
The present invention avoids many problems inherent in dynamic combinatorial chemistry. Library generation is performed in an organic medium that is immiscible with water and separated from the target molecule by a semipermeable membrane. The membrane isolates the target molecule within an aqueous phase by its size, yet allows small molecule ligand transport and diffusion. This approach affords several advantage. First, ligands are assembled in an organic phase, which provides a wide range of organic reactions and catalysts that are unsuccessful in water. Thus, chemical reactions which mutate the library are carried out in an environment which is isolated from the target molecule, thereby ensuring that the target molecule is not somehow chemically modified and bolstering confidence in the accuracy of binding assays. Second, in theory any reversible reaction can be employed. This allows for use of catalyzed forward and reverse reactions during assembly and disassembly of combinatorial products, as well as coordination chemistry of the type disclosed, for example, in U.S. patent application Ser. No. 09/181,108 to Miller et al., filed Oct. 28, 1998, which is hereby incorporated by reference in its entirety. Third, bimolecular reactions can be concentrated, guaranteeing a reaction rate that is faster than ligand-target molecule dissociation rate. This provides for affinity-based enrichment of the library products present in the aqueous phase.